# Novel Regulators of GPCR Signaling in Hematopoietic Stem Cells

> **NIH NIH F32** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 2021 · $68,562

## Abstract

Project Summary
Hematopoietic stem and progenitor cells (HSPCs) are self-renewing, transplantable cells that support lifelong
blood production. HSPC function and transplantation are tied to signal transduction through G-protein coupled
receptors (GPCRs), particularly that of CXCR4 which regulates homing and niche retention. Our understanding
of basic GPCR regulatory mechanisms in these cells are lacking. This is highlighted by our laboratories recent
finding that two GPCR-trafficking proteins, GPRASP1 and GPRASP2, act as negative regulators of HSPC
transplantation. Our preliminary data further indicate that these proteins limit HSPC quiescence and resistance
to apoptosis in a CXCR4-dependent manner. GPRASP1 and -2 have been described in the nervous system to
promote the lysosomal degradation and functional downregulation of opioid, dopamine, and other select
receptors. The receptors regulated by GPRASP1 and -2 in HSCs are currently unknown. As a postdoctoral fellow
in the McKinney-Freeman laboratory, I will investigate the molecular functions of GPRASP1 and -2 in HSPCs.
In Aim 1, I will use biochemical methods to definethe impact of GPRASP1 and -2 on CXCR4 signaling. The
impact of GPRASP1 and -2 on CXCR4 endocytosis, degradation, and downstream signaling outcomes after
agonist stimulation will be analyzed in mouse HSPCs. In Aim 2, I will probe the role of GPRASP1 and -2 in
transplantation of human HSPCs isolated from cord blood. Human HSPCs that have undergone gene editing by
CRISPR/Cas9 nucleofection to knockout GPRASP1 or -2 and will be used in competitive xenotransplantation
assays. By following the relative engraftment and blood production of wild type and knockout human HSPCs
after xenotransplantation the impact of GPRASP proteins on this procedure will be determined. In Aim 3, I will
use an unbiased proteomics approach to investigate the receptor targets of GPRASP1 and -2, as well as the
downstream trafficking pathways associated with these proteins. Biotin proximity labeling will be performed using
GPRASP-APEX fusion proteins in K562 chronic myelogenous leukemia cells both before and at multiple
timepoints following agonist stimulation. Biotinylated samples will undergo Tandem Mass Tagging and multiplex
mass spectrometry to determine the relative abundance of proteins across timepoints. This will allow for
bioinformatic analysis of the receptor targets and downstream trafficking pathways associated with GPRASP1
and -2. All protein associations of interest will be validated by immunofluorescence in mouse HSPCs. These
aims draw on my prior training as a biochemist but provide abundant training opportunities in the field of HSPC
biology. The McKinney-Freeman laboratory and St. Jude are ideal environments in which to receive training in
HSPC biology. Institutional resources provided, including state-of-the-art flow cytometry and proteomics cores,
will greatly contribute to the success of this project. In addition, I will take advantage of nume...

## Key facts

- **NIH application ID:** 10254425
- **Project number:** 5F32DK123993-03
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **Principal Investigator:** Christopher David Nevitt
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $68,562
- **Award type:** 5
- **Project period:** 2019-09-16 → 2022-09-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10254425

## Citation

> US National Institutes of Health, RePORTER application 10254425, Novel Regulators of GPCR Signaling in Hematopoietic Stem Cells (5F32DK123993-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10254425. Licensed CC0.

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